.The goal of this project is to elucidate structure-function relationships in macromolecular assemblies. During FY18, our studies focused on (1) retinoschisin (RS1), a junctional protein of the human retina; (2) encapsulin, a bacterial nanocompartment that sequesters iron; and (3) computational tools used in image analysis. 1) RS1 is a protein required to maintain the structural and functional integrity of the retina. Mutations in RS1 lead to early vision impairment in young males, a condition termed X-linked retinoschisis (XLRS). From prior work, RS1 was thought to form an octamer, with each subunit comprising a discoidin domain (DS) and a small N-terminal domain (RS1 domain). We used cryo-EM to determine the structure of RS1 at 0.4 nm resolution, finding that the complex actually consists, of two apposed octameric rings. The RS1 domains occupy the centers of the rings, but are less clearly defined, suggesting mobility. We combined the cryo-EM density map with crystal structures of other discoidin domains to create a high-resolution model of the double octamer. This model is consistent with known intramolecular and intermolecular disulfide bonds. The interfaces between subunits in a ring and between rings accommodate residues mutated in some XLRS patients. Their placement indicates the importance of correct assembly of the 16-mer complex to obtain a viable junction. These results were published in 2016. Some of the remaining XLRS-related mutations map to spike features at the periphery of the rings. Based on biochemical data, RS1 has been proposed to interact with cell membranes by binding to lipid head-groups and/or carbohydrate moieties on glycolipids or glycoproteins. In particular, RS1 has been shown to bind to galactose. To observe its effect, we performed cryo-EM on RS1 with galactose bound. Under these conditions, RS1 assembles into long linear strands with branches. The molecules in these aggregates show significant deviations from symmetry, suggesting some flexibility. To visualize the deviations, we performed asymmetric reconstructions, identifying four types of interaction. The first is within a strand, where there are strong spike-spike interactions between adjacent molecules. The second is an arrangement whereby four strands emanate from a central molecule. The third and four types involve sideways interactions with further branching. The ability of RS1 to form multi-strand networks raises the possibility that in vivo it may form a three-dimensional scaffold between photoreceptor cells to glue them together. The latter observations. together with a generalized junctional model, are summarized in a paper submitted for publication. 2) Encapsulins are protein shells that resemble viral capsids in many respects, including the fold of their constituent subunits and the icosahedral symmetry of their molecular architecture. This fold was first observed in capsids of bacteriophage HK97. However, instead of housing genomic nucleic acid as in viral capsids, encapsulins accommodate other kinds of cargo. In a paper published in FY15, we described the structure and essential functional properties of an encapsulin of the Gram-negative bacterium Myxococcus xanthus. This particle has an icosahedral protein shell 32 nm in diameter and an icosahedral triangulation number of 3. Thus EncA is assembled from 180 copies of EncA protein; it also has smaller amounts of three internal proteins (EncB; EncC; EncD). Native nanocompartments isolated from M. xanthus have dense iron-rich cores. Functionally, they resemble ferritins, but with a massively greater capacity (30,000 Fe atoms vs. 3,000 in ferritin). Their role appears to be to scavenge excess cytoplasmic iron in order to protect the bacterium from reactive oxygen species. In continuing studies on this system, our main thrust has been to seek high resolution cryo-EM data on the purified cargo protein ClpB. ClpB has a disordered C-terminal region but its N-terminal regions is folded and forms a decamer with D5 symmetry. In our current model for the complex, these decamers are envisaged to bind to specific sites on the inner surface of EncA shells whereby aligned channels through the respective walls of the EncA and the EncB shells allow iron and lesser amounts of phosphorus to enter the EncB shell where they are deposited. 3) Development of image processing software for three-dimensional electron microscopy. Bsoft is a comprehensive suite of computer programs for image processing of cryo-EM images and cryo-ET data that is maintained, disseminated, and further developed in the LSBR by B. Heymann. Over FY17 and FY18, an updated and upgraded version of Bsoft (Bsoft 2.0.0) was released. In it, the code structure was modified to eliminate legacy libraries and to introduce a more general compilation scheme. The intention is to develop more in alignment with modern C++ standards to ensure better stability and longevity. The single particle analysis (SPA) capabilities have been expanded to allow better 2D analysis and classification. Specifically, the handling of dose-fractionated movies (motion correction) has been improved; the processing of tomographic tilt series can now be done without the need to write command lines. This includes estimation of the contrast transfer function parameters and correcting for the CTF during reconstruction. In the last few years, advances both in cryo-microscopy per se and in image processing have made it possible to reconstruct density maps at resolutions as high as those achieved by X-ray crystallography. This development has raised the question of whether any particular work-flows or processing strategies achieve the best results and/or whether artifacts may be introduced in some circumstances. To this end, B. Heymann has been participating, together with computational specialists from other institutions in the Map Challenge project, both in submitting two reconstructions obtained with his Bsoft package and in writing up several conclusions emerging from the project.